P. aeruginosa is one of the principal pathogens associated with Cystic fibrosis (CF) pulmonary infection and chronic and burn wounds. Once established, P. aeruginosa biofilms are difficult to eradicate by antimicrobial treatment. The nature of biofilm resistance has been deemed multifactorial. However, recent findings in our laboratory challenge the current dogma and suggest instead the existence of a classical, biofilm-specific mechanism of resistance in P. aeruginosa. Our data suggest that biofilm resistance is regulated by the biofilm- specific transcriptional regulator BrlA (PA4878), a member of the MerR family of transcriptional regulators which activate expression of multi drug transporters upon binding of the transporter substrate. The goal of the proposed study is to determine the mechanism by which BrlA regulates antimicrobial resistance of P. aeruginosa biofilms. Experimentally, we will first determine why brlA is only expressed under biofilm growth conditions. We hypothesize that physicochemical gradients and growth conditions to which bacteria are subjected to in biofilms, are responsible for activating brlA gene expression. We will make use of brlA-reporter gene fusions and expose reporter strains grown planktonically to "biofilm-like" growth condition. Induction of brlA gene expression will be monitored via fluorescence. Furthermore, transposon mutagenesis will be used to identify proteins that repress/activate brlA under planktonic or biofilm growth conditions, respectively. Based on our preliminary data, we hypothesize that BrlA is a global transcriptional regulator of multidrug efflux pump genes (mexAB-oprM, mexGHI-opmD, mexEF-oprN, and oprH-phoPQ). To determine whether BrlA acts as transcriptional activator and binds to the promoters of brlA, oprH, mexE, phoP, and mexA, gel mobility shift and DNAse I footprinting assays will be used in Specific Aim 2. Genes/promoters for which BrlA-binding has been confirmed will be further analyzed for BrlA-dependent gene expression in biofilms and biofilm-like condition using gfp reporter gene fusions. Based on our preliminary data, we anticipate analyzing the expression of mexAB-oprM and mexEF-oprN. To establish a firm link between BrlA, expression of multidrug efflux pump genes, and biofilm resistance, CFU counts and biofilm MIC testing will be carried out in Specific Aim 3 to determine biofilm resistance of isogenic mutants of mexAB-oprM and mexEF-oprN. In case none of the mex mutants are as susceptible as brlA biofilms, we will quantitatively compare the membrane protein composition of biofilms by P. aeruginosa PAO1, brlA mutant biofilms (susceptible), and mex mutant biofilms showing intermediate resistance, and identify membrane proteins that are differentially produced in susceptible/resistant biofilms that may contribute to biofilm resistance. Findings from this detailed investigation are expected to elucidate the mechanism of BrlA-dependent regulation of P. aeruginosa biofilm resistance and eventually lead to innovative and more effective treatment strategies based on inhibition or regulation of biofilm resistance to treat and control biofilm infections. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is a common cause of hospital acquired infection and the leading cause of death in patients with cystic fibrosis. One of the hallmarks of P. aeruginosa is its high intrinsic resistance to antibiotics. This proposal is aimed at understanding the mechanism of biofilm resistance and the role of the novel transcriptional regulator BrlA in regulating antimicrobial resistance of P. aeruginosa biofilms. Findings from this research may lead to novel and innovative treatment strategies to treat and eradicate biofilm infections.